The vascular endothelium responds to localized infection by weakening barrier function and promoting coagulation to deliver humoral effectors of immunity and contain the spread of microbes. In systemic infection, these same protective responses become maladaptive, leading to diffuse vascular leakage and disseminated intravascular coagulation. We hypothesize that the septic endothelium drives critical disease manifestations, and we seek innovative ways to target and measure this complex process. Aim 1 will evaluate the roles of two major endothelial cell-surface receptors, PAR1 and Tie2, whose activation can promote inflammation, thrombosis, and barrier dysfunction during sepsis. We have identified novel drug-like molecules that target PAR1 and Tie2 and synergize to achieve endothelial quiescence in the face of septic stimuli. This aim will ask how PAR1 and Tie2 collaborate at the endothelium to ameliorate clinically relevant outcomes of sepsis. Aim 2 will address how the endothelium contributes to septic microthromboses. In addition to evaluating PAR1 and Tie2, this aim will provide a first-in-kind systematic description of the endothelial derangements that drive the early microthromboses and late coagulopathy of sepsis by utilizing a combination of intravital microscopy in genetic mouse models and novel cell-based approaches. Aim 3 will seek new non-invasive ways to follow the dynamic responses of the endothelium to sepsis. We have developed quantitative, real-time, humanized assays of microvascular barrier dysfunction and endothelial prothrombotic potential suitable for high-throughput analysis. Applying plasmas from one of the largest ICU cohorts of its kind, we will test the predictive performance of these assays against clinical metrics and conventional biomarkers. We will also explore the potential of these assays to gauge the efficacy of new candidate therapies. This application unites a team of investigators with complementary expertise, unique resources, and a track record of productivity and collaboration. We will pursue highly parallel aims designed to generate outstanding impact in sepsis by defining fundamental disease mechanisms, testing innovative therapeutic strategies, and developing robust new diagnostic tools focused on the endothelium.